Method to regulate CD40 signaling

ABSTRACT

The present invention relates to methods useful for identifying compounds capable of specifically controlling CD40 regulation of JNK or p38 activity useful for inhibiting immunoglobulin heavy chain class switching, cytokine production and activation of cells involved in an inflammatory response. The present invention also includes kits to perform such assays and methods to control disease related to such responses.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority from U.S. ProvisionalApplication Serail No. 60/008,877, filed Dec. 19, 1995.

GOVERNMENT RIGHTS

[0002] This invention was made in part with government support underDK-37871 and GM-30324, both awarded by the National Institutes ofHealth. The government has certain rights to this invention.

FIELD OF THE INVENTION

[0003] The present invention relates to a process for identifyingcompounds that control CD40 regulation of Jun kinase and p38 MAP kinaseactivity. The present invention also relates to a method to treatallergies and autoimmunity by regulating the activity of Jun kinase andp38 MAP kinase in a cell.

BACKGROUND OF THE INVENTION

[0004] The B lymphocyte surface antigen receptor membrane immunoglobulinhas important functions in the binding and internalization of antigen,as well as in transducing signals through the plasma membrane which leadto cell activation, differentiation and apoptosis. Crosslinking of thereceptor stimulates the Ras/Raf-1/MEK cascade with activation ofp42^(erk2) MAPK and p90^(rsk). A second important B cell surface antigenreceptor is CD40. CD40 is a 45-50 kD transmembrane glycoproteinexpressed on all mature B cells. CD40 is a member of the TNF receptorfamily and has homology to the receptors for nerve growth factor, TNF-α,Fas and CD30. The ligand for CD40 (CD40L, gp39) is expressed onactivated T lymphocytes and activation through CD40 plays an importantrole in T cell-dependent immunoglobulin isotype switching. The signaltransduction pathways through CD40 are not well delineated.

[0005] Certain biological functions of a B lymphocyte (B cell) aretightly regulated by signal transduction pathways within B cells. Signaltransduction pathways maintain the balanced steady state functioning ofa cell. Disease states can arise when the steady state function of acell becomes harmful to an animal. For example, allergic reactions occurdue to undesired production of IgE antibodies specific for an antigen.In addition, autoimmunity can occur due to an animal mounting anundesired immune response against a self-antigen. Signal transductionpathways in a cell can be responsible for regulating cellular biologicalfunctions. As such, regulation of signal transduction pathways canassist in the regulation of undesired cellular biological functions.

[0006] Despite a long-felt need to understand and discover methods forregulating cells involved in various disease states, the complexity ofsignal transduction pathways has precluded the development of productsand processes for regulating cellular function by manipulating signaltransduction pathways in a cell. As such, there remains a need forproducts and processes that permit the implementation of predictablecontrols of signal transduction in cells, thus enabling the treatment ofvarious diseases that are caused by undesired cellular function.

SUMMARY OF THE INVENTION

[0007] The present invention provides a solution to the complex problemof identifying regulatory compounds which can be used to regulatecellular function, including CD40 regulation of Jun kinase (JNK) and p38MAP kinase (p38) activity. Despite the complexity of signal transductionnetworks in cells, the present invention provides for an efficientmethod for regulating JNK or p38 activity and identifying compoundscapable of specifically regulating JNK or p38 activity.

[0008] The present invention is particularly advantageous because itprovides for a method to identify compounds that can regulate theproduction of IgE by an animal without substantially interfering withthe production of IgG and IgA by an animal. As such, unlike traditionalimmunosuppressive reagents which suppress an animal's immune responseindiscriminately, a compound identified by a method of the presentinvention enables an animal to mount an immune response against foreignpathogens by producing IgG and IgA antibodies. Thus, those of skill inthe art will immediately recognize the advantages arising from thisinvention which include the identification and uses of compounds whichare useful for the treatment of allergic and autoimmune diseases but notdisruptive to an animal's overall immune response.

[0009] One embodiment of the present invention includes a method toidentify a compound that controls CD40 regulation of Jun kinase (JNK)activity in a cell, comprising: (1) contacting a cell with a putativeregulatory compound, wherein the cell includes a CD40 protein and a Junkinase protein; and (2) assessing the ability of the putative regulatorycompound to regulate the activity of the Jun kinase. Another embodimentof the present invention includes a method to identify a compound thatcontrols CD40 regulation of p38 activity in a cell, comprising: (1)contacting a cell with a putative regulatory compound, wherein the cellincludes CD40 protein and p38 protein; and (2) assessing the ability ofthe putative regulatory compound to regulate the activity of the p38protein. In particular, these methods of the present invention include astep of stimulating the cell, prior to the assessing step, with a ligandof CD40.

[0010] Also included in the present invention is a regulatory compoundidentified by said compound's ability to regulate a biological functionselected from the group consisting of immunoglobulin heavy chain classswitching, cytokine production and inflammatory cell activation, thecompound being capable of penetrating the plasma membrane of a cell andof inhibiting the ability of CD40 protein to regulate JNK protein or p38activity in the cell.

[0011] The present invention includes a method to inhibit immunoglobulinheavy chain class switching, comprising inhibiting the activity of aprotein selected from the group consisting of Jun kinase protein and p38protein. The present invention also includes a method to inhibitcytokine production by a cell having CD40, comprising inhibiting theactivity of a protein selected from the group consisting of Jun kinaseprotein and p38 protein.

[0012] One aspect of the present invention includes a method to treat ananimal with a disease selected from the group consisting of a diseaseinvolving an allergic response and an autoimmune disease, said methodcomprising administering to an animal an effective amount of atherapeutic composition comprising a compound that controls CD40regulation of the activity of a protein selected from the groupconsisting of Jun kinase and p38 protein.

[0013] Another aspect of the present invention includes a kit toidentify compound that controls CD40 regulation of JNK or p38 activityin a cell, the kit comprising: (1) a cell comprising a CD40 protein, anda Jun kinase or a p38 protein; and (2) a means for detecting regulationof the Jun kinase or p38 protein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 illustrates the activation of ERK protein followingtreatment with anti-IgM antibody but not after treatment with anti-CD40antibody.

[0015]FIG. 2 illustrates the time course of JNK activation aftertreatment of Ramos cells with 1 μg/ml anti-CD40 antibody.

[0016]FIG. 3 illustrates the dose response of anti-CD40 antibodyactivated JNK in Ramos cells treated using various concentrations ofanti-CD40 antibody for 15 min.

[0017]FIG. 4 illustrates the dose response of JNK activity to anti-CD40antibody stimulation in tonsillar B cells treated for 15 min.

[0018]FIG. 5 illustrates activation of JNK using soluble gp39, in whichdilutions of culture supernatants containing soluble gp39 were added toRamos cells for 15 min.

[0019]FIG. 6 illustrates the absence of stimulation of JNK activity inRamos cells by anti-IgM antibody at different time periods.

[0020]FIG. 7 illustrates the absence of stimulation of JNK activity intonsillar B cells at different time periods.

[0021]FIG. 8 illustrates the activation of Ras following treatment ofcells with anti-IgM antibody but not after treatment with anti-CD40antibody.

[0022]FIG. 9 illustrates the activation of MEKK protein followingtreatment with anti-CD40 antibody.

[0023]FIG. 10 illustrates the activation of JNK in Ramos cells byanti-IgM or anti-CD40 antibody alone, or after preincubation withanti-CD40 antibody followed by incubation with anti-IgM antibody.

[0024]FIG. 11 illustrates activation of p38 in the presence or absenceof anti-CD40 antibody.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention relates to a method for identifyingcompounds that regulate CD40 regulation of JNK or p38 activity andproducts identified using such method. As used herein, the phrase“signal transduction pathway” refers to at least one biochemicalreaction, but more commonly a series of biochemical reactions, whichresult from interaction of a cell with a stimulatory compound. Theinteraction of a stimulatory compound with a cell generates a “signal”that is transmitted through a signal transduction pathway, ultimatelyresulting in JNK or p38 activation. Compounds inhibitory to signaltransduction pathways (antagonists) are also useful and can beidentified by the methods of the present invention.

[0026] A signal transduction pathway of the present invention caninclude a variety of signal transduction molecules that play a role inthe transmission of a signal from one portion of a cell to anotherportion of a cell. As used herein, the term “molecule” refers to aprotein, a lipid, a nucleic acid or an ion, and at times is usedinterchangeably with such terms. In particular, a signal transductionmolecule refers to a protein, a lipid, a nucleotide, or an ion involvedin a signal transduction pathway. Signal transduction molecules of thepresent invention include, for example, cell surface receptors andintracellular signal transduction molecules. As used herein, the phrase“cell surface receptor” includes molecules and complexes of moleculescapable of receiving a signal and the transmission of such a signalacross the plasma membrane of a cell. The phrase “intracellular signaltransduction molecule,” as used herein, includes those molecules orcomplexes of molecules involved in transmitting a signal from the plasmamembrane of a cell through the cytoplasm of the cell, and in someinstances, into the cell's nucleus. The phrase “stimulatory compound”,as used herein, includes ligands capable of binding to cell surfacereceptors to initiate a signal transduction pathway, as well asintracellular initiator molecules capable of initiating a signaltransduction pathway from inside a cell. One aspect of the presentinvention includes a cell-based assay to identify compounds, referred toherein as “putative regulatory compounds”, which are capable ofregulating CD40 regulation of JNK or p38 activity. As used herein, theterm “putative” refers to compounds having an unknown or previouslyunappreciated regulatory activity in a particular process. As such, theterm “identify” is intended to include all compounds, the usefulness ofwhich as a regulatory compound of JNK activity is determined by a methodof the present invention.

[0027] One embodiment of the present invention relates to a method toidentify a compound that controls CD40 regulation of JNK activity in acell, comprising: (1) contacting a cell with a putative regulatorycompound, wherein the cell includes a CD40 protein and a Jun kinase(JNK) protein; and (2) assessing the ability of the putative regulatorycompound to regulate the activity of the Jun kinase. The assessment steppreferably involves determining the phosphorylation of JNK upon ligationof the CD40 using antibodies specific for CD40 and/or CD40 ligand (gp39;described in Noelle et al., Proc. Natl. Acad. Sci. USA 89:6550-6554,1992). JNK regulates the activity of the transcription factor JUN whichis involved in controlling the growth and differentiation of differentcell types, such as B cells, T cells, neural cells or fibroblasts. JNKis a member of the Ras signal transduction pathway, which includesmolecules such as MEKK protein Jun, ATF and Myc protein.

[0028] Another embodiment of the present invention relates to a methodto identify a compound that controls CD40 regulation of p38 activity ina cell, comprising: (1) contacting a cell with a putative regulatorycompound, wherein the cell includes a CD40 protein and a p38 protein;and (2) assessing the ability of the putative regulatory compound toregulate the activity of the p38.

[0029] The present method can further comprise assessing the ability ofa putative regulatory compound to inhibit: immunoglobulin heavy chainclass switching in a cell; cytokine production by a cell; or activationof inflammatory cells (i.e., cells involved in an inflammatoryresponse). Methods for determining immunoglobulin heavy chain classswitching are to those of skill in the art. For example, Southern blotscan be performed using DNA probes specific for genes encoding differentclasses of immunoglobulin heavy chains to look for rearrangement of theDNA encoding the different classes. Alternatively, immunoassays can beperformed on proteins produced by the treated cell using antibodiesspecific for different classes of immunoglobulin heavy chains. Methodsfor determining cytokine production are known to those of skill in theart. For example, cell responsiveness assays using cells capable ofresponding to a cytokine can be used to test the disruption of cytokineproduction by a putative regulatory compound. In addition, immunoassaysusing antibodies specific for a cytokine can be used to test thedisruption of cytokine production by a putative regulatory compound.Methods for determining inhibition of inflammatory cell activation areknown to those of skill in the art by testing the ability of aninflammatory cell to perform a desired biological function in thepresence or absence of a putative regulatory protein.

[0030] Suitable cells for use with the present invention include anycell that has CD40, and JNK or p38 protein. Such cells can includenormal cells or transformed cells (i.e., with a heterologous nucleicacid molecule) that express CD40, and JNK and/or p38 in a nativephysiological context (e.g., Pre-B cells, B lymphocytes, cancer cells,fibroblasts, Langerhans cells, epithelial cells monocytes and dendriticcells). Alternatively, cells for use with the present invention caninclude spontaneously occurring variants of normal cells, or geneticallyengineered cells, that have altered signal transduction activity, suchas enhanced responses to particular ligands. Signal transductionvariants of normal cells can be identified using methods known to thosein the art. For example, variants can be selected using fluorescenceactivated cell sorting (FACS) based on the level of calcium mobilizationby a cell in response to a ligand. Genetically engineered cells caninclude recombinant cells of the present invention (described in detailbelow) that have been transformed with, for example, a recombinantmolecule encoding a signal transduction molecule of the presentinvention.

[0031] Cells for use with the present invention include mammalian,invertebrate, plant, insect, fungal, yeast and bacterial cells.Preferred cells include mammalian, amphibian and yeast cells. Preferredmammalian cells include primate, non-human primate, mouse and rat, withhuman cells being preferred.

[0032] In one embodiment, a cell suitable for use in the presentinvention has a functional CD40 on the surface of the cell. A functionalCD40 can comprise a full-length or a portion of a CD40 that is capableof transmitting a signal across the plasma membrane of a cell, uponligation with an anti-CD40 antibody or a CD40 ligand, in such a mannerthat immunoglobulin heavy chain class switching results. Preferably, acell of the present invention expresses a CD40 derived from a human,mouse or rat, with human cells being preferred.

[0033] In another embodiment, a cell suitable for use in the presentinvention has one or more intracellular signal transduction moleculescapable of transmitting a signal through the cytoplasm of the cell,resulting in JNK and/or p38 activation. An intracellular signaltransduction molecule as described herein can be produced in a cell byexpression of a naturally occurring gene and/or by expression of aheterologous nucleic acid molecule transformed into the cell.

[0034] A preferred cell of the present invention has, amongst othersignal transduction molecules, MEKK protein, Jun, ATF Myc protein, p38,phosphotidylinositol-3 kinase (PI-3 kinase), Jun kinase kinase (JNKK),Elk-1 and other Ets family members, phospholipase C γ (PLCγ) andintracellular calcium.

[0035] In a preferred embodiment, a cell for use with the presentinvention includes the human Burkitt's lymphoma cell line, Ramos.

[0036] Signal transduction molecules referred to herein include thenatural full-length protein, or can be a functionally equivalent proteinin which amino acids have been deleted (e.g., a truncated version of theprotein), inserted, inverted, substituted and/or derivatized (e.g.,phosphorylated, acetylated, glycosylated, carboxymethylated,myristoylated, prenylated or palmitoylated amino acids) such that themodified protein has a biological activity and/or function substantiallysimilar to that of the natural protein. Modifications can beaccomplished by techniques known in the art, including, but not limitedto, direct modifications to the protein or modifications to the geneencoding the protein. Such modifications to the gene encoding theprotein can include using, for example, classic or recombinant DNAtechniques to effect random or targeted mutagenesis (see, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Labs Press, 1989, which is incorporated herein by reference inits entirety).

[0037] Functionally equivalent proteins can be selected using assaysestablished to measure the biological activity of the protein. Forexample, a functionally equivalent cell surface receptor would have asimilar ability to bind a particular ligand as would the correspondingnatural cell surface receptor protein. As a further example, afunctionally equivalent intracellular signal transduction protein wouldhave a similar ability to associate with and regulate the activity ofanother intracellular molecule as would the corresponding naturalintracellular signal transduction protein.

[0038] In certain embodiments, a cell of the present invention istransformed with at least one heterologous nucleic acid molecule. Anucleic acid molecule as described herein can be DNA, RNA, or hybrids orderivatives of either DNA or RNA. Nucleic acid molecules as referred toherein can include regulatory regions that control expression of thenucleic acid molecule (e.g., transcription or translation controlregions), full-length or partial coding regions, and combinationsthereof. It is to be understood that any portion of a nucleic acidmolecule can be produced by: (1) isolating the molecule from its naturalmilieu; (2) using recombinant DNA technology (e.g., PCR amplification,cloning); or (3) using chemical synthesis methods. A gene includes allnucleic acid sequences related to a natural cell surface receptor genesuch as regulatory regions that control production of a cell surfacereceptor encoded by that gene (such as, but not limited to,transcription, translation or post-translation control regions) as wellas the coding region itself.

[0039] A nucleic acid molecule can include functional equivalents ofnatural nucleic acid molecules encoding a protein. Functionalequivalents of natural nucleic acid molecules can include, but are notlimited to, natural allelic variants and modified nucleic acid moleculesin which nucleotides have been inserted, deleted, substituted, and/orinverted in such a manner that such modifications do not substantiallyinterfere with the nucleic acid molecule's ability to encode a moleculeof the present invention. Preferred functional equivalents includesequences capable of hybridizing under stringent conditions (i.e.,sequences having at least about 70% identity), to at least a portion ofa signal transduction protein encoding nucleic acid molecule accordingto conditions described in Sambrook et al., ibid.

[0040] As guidance in determining what particular modifications can bemade to any particular nucleic acid molecule, one of skill in the artshould consider several factors that, without the need for undueexperimentation, permit a skilled artisan to appreciate workableembodiments of the present invention. For example, such factors includemodifications to nucleic acid molecules done in a manner so as tomaintain particular functional regions of the encoded proteinsincluding, a ligand binding site, a target binding site, a kinasecatalytic domain, etc. Functional tests for these variouscharacteristics (e.g., ligand binding studies and signal transductionassays such as kinase assays, and other assays described in detailherein and those known by those in the art) allows one of skill in theart to determine what modifications to nucleic acid sequences would beappropriate and which would not.

[0041] Transformation of a heterologous nucleic acid molecule (e.g., aheterologous cell surface receptor encoding a nucleic acid molecule)into a cell suitable for use in the present invention can beaccomplished by any method by which a gene is inserted into a cell.Transformation techniques include, but are not limited to, transfection,retroviral infection, electroporation, lipofection, bacterial transferand spheroplast fusion. Nucleic acid molecules transformed into cellssuitable for use in the present invention can either remain onextra-chromosomal vectors or can be integrated into the cell genome.

[0042] Expression of a nucleic acid molecule of the present invention ina cell can be accomplished using techniques known to those skilled inthe art. Briefly, the nucleic acid molecule is inserted into anexpression vector in such a manner that the nucleic acid molecule isoperatively joined to a transcription control sequence in order to becapable of effecting either constitutive or regulated expression of thegene when the gene is transformed into a host cell. Construction ofdesired expression vectors can be performed by methods known to thoseskilled in the art and expression can be in eukaryotic or prokaryoticsystems An expression system can be constructed from control elements,including transcription control sequences, translation controlsequences, origins of replication, and other regulatory sequences thatare compatible with a host cell, operatively linked to nucleic acidsequences using methods known to those of skill in the art. (see, forexample, Sambrook et al., ibid. ).

[0043] In one embodiment, a cell suitable for use in the presentinvention is transformed with a nucleic acid molecule encoding CD40, JNKand/or p38, as described in detail herein. In another embodiment of thepresent invention, a cell suitable for use in the present invention istransformed with a nucleic acid molecules encoding at least one type ofintracellular signal transduction protein of the present invention.Preferred intracellular signal transduction protein encoding nucleicacid molecules include, but are not limited to, nucleic acid moleculesencoding JNK, p38, MEKK, Jun, ATF, Myc protein, p38, PI-3 kinase, CDC42,Rho, Rac, JAK family of kinases (e.g. JAK1, JAK2, JAK3), STAT family ofkinases, JNKK, Elk-1 and other Ets family members, PLCγ andintracellular calcium.

[0044] It is within the scope of the present invention that a cell canbe transformed with both a nucleic acid molecule encoding at least onetype of signal transduction molecule and a nucleic acid moleculeencoding at least one type of cell surface receptor.

[0045] In one embodiment, the method of the present invention comprisescontacting a cell with a putative regulatory compound. According to thepresent invention, putative regulatory compounds include compounds thatare suspected of being capable of regulating CD40, JNK and/or p38activity. The term “activity” refers to any stage of activation of asignal transduction molecule by, for example, conformational change of amolecule which results in the acquisition of catalytic activity by themolecule; the phosphorylation of a molecule, thereby resulting in theacquisition or loss of catalytic activity by the molecule; or thetranslocation of a molecule from one region of a cell to another,thereby enabling the molecule to bind another molecule. The term“regulate” refers to controlling the activity of a molecule and/orbiological function, such as enhancing or diminishing such activity orfunction.

[0046] Putative compounds as referred to herein include, for example,compounds that are products of rational drug design, natural productsand compounds having partially defined signal transduction regulatoryproperties. A putative compound can be a protein-based compound, acarbohydrate-based compound, a lipid-based compound, a nucleicacid-based compound, a natural organic compound, a synthetically derivedorganic compound, an anti-idiotypic antibody and/or catalytic antibody,or fragments thereof. A putative regulatory compound can be obtained,for example, from libraries of natural or synthetic compounds, inparticular from chemical or combinatorial libraries (i.e., libraries ofcompounds that differ in sequence or size but that have the samebuilding blocks; see for example, U.S. Pat. Nos. 5,010,175 and 5,266,684of Rutter and Santi, which are incorporated herein by reference in theirentirety) or by rational drug design.

[0047] In a rational drug design procedure, the three-dimensionalstructure of a compound, such as a signal transduction molecule can beanalyzed by, for example, nuclear magnetic resonance (NMR) or x-raycrystallography. This three-dimensional structure can then be used topredict structures of potential compounds, such as putative regulatorycompounds by, for example, computer modelling. The predicted compoundstructure can then be produced by, for example, chemical synthesis,recombinant DNA technology, or by isolating a mimetope from a naturalsource (e.g., plants, animals, bacteria and fungi). Potential regulatorycompounds can also be identified using SELEX technology as described in,for example, PCT Publication Nos. WO 91/19813; WO 92/02536 and WO93/03172 (which are incorporated herein by reference in their entirety).

[0048] In particular, a naturally-occurring intracellular signaltransduction molecule can be modified based on an analysis of itsstructure and function to form a suitable regulatory compound. Forexample, a compound capable of regulating the kinase domain of JNK cancomprise a compound having similar structure to a residues 1-79 of theamino terminus of JNK. Such a compound can comprise a peptide, apolypeptide or a small organic molecule.

[0049] Putative regulatory compounds can also include molecules designedto interfere with CD40. For example, mutant CD40 can be created thatinterfere with the coupling of the receptor to intracellular signaltransduction proteins. Alternatively, mutant CD40 can be created thatinterfere with the binding of CD40 ligand to CD40. Putative regulatorycompounds can include agonists and antagonists of CD40. Such agonistsand antagonists can be selected based on the structure of anaturally-occurring ligand to CD40.

[0050] The conditions under which the cell of the present invention iscontacted with a putative regulatory compound, such as by mixing, areconditions in which the cell can exhibit JNK and/or p38 activity ifessentially no other regulatory compounds are present that wouldinterfere with such activity. Achieving such conditions is within theskill in the art, and includes an effective medium in which the cell canbe cultured such that the cell can exhibit JNK and/or p38 activity. Forexample, for a mammalian cell, effective media are typically aqueousmedia comprising RPMI 1640 medium containing 10% fetal calf serum.

[0051] Cells of the present invention can be cultured in a variety ofcontainers including, but not limited to, tissue culture flasks, testtubes, microtiter dishes, and petri plates. Culturing is carried out ata temperature, pH and carbon dioxide content appropriate for the cell.Such culturing conditions are also within the skill in the art. Forexample, for Ramos cells, culturing can be carried out at 37° C., in a5% CO₂ environment.

[0052] Acceptable protocols to contact a cell with a putative regulatorycompound in an effective manner include the number of cells percontainer contacted, the concentration of putative regulatorycompound(s) administered to a cell, the incubation time of the putativeregulatory compound with the cell, the concentration of ligand and/orintracellular initiator molecules administered to a cell, and theincubation time of the ligand and/or intracellular initiator moleculewith the cell. Determination of such protocols can be accomplished bythose skilled in the art based on variables such as the size of thecontainer, the volume of liquid in the container, the type of cell beingtested and the chemical composition of the putative regulatory compound(i.e., size, charge etc.) being tested.

[0053] In one embodiment of the method of the present invention, asuitable number of cells are added to a 96-well tissue culture dish inculture medium. A preferred number of cells includes a number of cellsthat enables one to detect a change in JNK activity using a detectionmethod of the present invention (described in detail below). A morepreferred number of cells includes between about 1 and 1×10⁶ cells perwell of a 96-well tissue culture dish. Following addition of the cellsto the tissue culture dish, the cells can be preincubated at 37° C., 5%C₂O for between about 0 to about 24 hours.

[0054] A suitable amount of putative regulatory compound(s) suspended inculture medium is added to the cells that is sufficient to regulate theactivity of a CD40, JNK and/or p38 protein in a cell such that theregulation is detectable using a detection method of the presentinvention. A preferred amount of putative regulatory compound(s)comprises between about 1 nM to about 10 mM of putative regulatorycompound(s) per well of a 96-well plate. The cells are allowed toincubate for a suitable length of time to allow the putative regulatorycompound to enter a cell and interact with a signal transductionmolecule. A preferred incubation time is between about 1 minute to about48 hours.

[0055] In another embodiment of the method of the present invention,cells suitable for use in the present invention are stimulated with astimulatory molecules capable of binding to CD40 of the presentinvention to initiate a signal transduction pathway and create acellular response. Preferably, cells are stimulated with a stimulatorymolecule following contact of a putative regulatory compound with acell. Suitable stimulatory molecules can include, for example,antibodies that bind specifically to the extracellular domain of CD40and CD40 ligand. Preferred stimulatory molecules include, but are notlimited to, anti-human CD40 antibody G28-5, soluble gp39, membrane-boundgp39 (e.g. gp39 bound to the plasma membrane of a cell or gp39incorporated into a synthetic lipid-based substrate such as a liposomeor micelle) and mixtures thereof. A suitable amount of stimulatorymolecule to add to a cell depends upon factors such as the type ofligand used (e.g., monomeric or multimeric; permeability, etc.) and theabundance of the receptor on a cell. Preferably, between about 1.0 nMand about 1 mM of ligand is added to a cell.

[0056] The method of the present invention include determining if aputative regulatory compound is capable of regulating JNK activation.Such methods include assays described in detail in the Examples section.The method of the present invention can further include the step ofperforming a toxicity test to determine the toxicity of a putativeregulatory compound.

[0057] Another aspect of the present invention includes a kit toidentify compounds capable of regulating CD40 regulation of JNK or p38activity in a cell. Such a kit includes: (1) a cell comprising CD40protein, and JNK and/or p38 protein; and (2) a means for detectingregulation of either the JNK or p38 protein. Such a means for detectingthe regulation of JNK protein include methods and reagents known tothose of skill in the art, for example, JNK activity can be detectedusing, for example, activation assays described in Example 2. Means fordetecting the regulation of p38 protein also include methods andreagents known to those of skill in the art. Suitable cells for use witha kit of the present invention include cells described in detail herein.A preferred cell for use with a kit includes, a human cell.

[0058] The present invention also includes the determination as towhether a putative regulatory compound is capable of regulating abiological response in a mammal. Such a method entails administering aputative regulatory compound to an animal, such compound being shown,using an assay of the present invention, to regulate CD40, JNK and/orp38 activity in a cell. Such a determination is useful for determiningconditions under which a putative regulatory compound can beadministered to an animal as a therapeutic composition. Thus, it iswithin the scope of the present invention that those conditions statedherein for testing a compound in an animal can be used whenadministering a therapeutic composition of the present invention. Inparticular, a putative regulatory compound can be administered to ananimal to determine if the compound is capable of regulating, forexample, an inflammatory response, a response to an infectious agent, anautoimmune response, a metabolic response, a cardiovascular response, anallergic response and/or an abnormal cellular growth response in theanimal. Acceptable protocols to administer putative regulatory compoundsto test the effectiveness of the compound include individual dose size,number of doses, frequency of dose administration, and mode ofadministration. Determination of such protocols can be accomplished bythose skilled in the art. A suitable single dose is a dose that iscapable of altering a biological response in an animal when administeredone or more times over a suitable time period (e.g., from minutes todays or weeks). Preferably, a dose comprises from about 1 nanogram ofthe compound per kilogram of body weight (ng/kg) to about 1 gram ofcompound per kilogram of body weight (gm/kg), more preferably 100 ng/kgto about 100 milligrams/kilogram (mg/kg), and even more preferably fromabout 10 micrograms of compound per kilogram of body weight to about 10mg/kg. Modes of administration can include, but are not limited to,aerosolized, subcutaneous, rectally, intradermal, intravenous, nasal,oral, transdermal and intramuscular routes. A putative regulatorycompound can be combined with other components such as apharmaceutically acceptable excipient and/or a carrier, prior toadministration to an animal. Examples of such excipients include water,saline, Ringer's solution, dextrose solution, Hank's solution, and otheraqueous physiologically balanced salt solutions. Nonaqueous vehicles,such as fixed oils, sesame oil, ethyl oleate, or triglycerides may alsobe used. Other useful formulations include suspensions containingviscosity enhancing agents, such as sodium carboxymethylcellulose,sorbitol, or dextran. Excipients can also contain minor amounts ofadditives, such as substances that enhance isotonicity and chemicalstability. Examples of buffers include phosphate buffer, bicarbonatebuffer and Tris buffer, while examples of preservatives includethimerosal, m- or o-cresol, formalin and benzyl alcohol. Standardformulations can either be liquid injectables or solids which can betaken up in a suitable liquid as a suspension or solution for injection.Carriers are typically compounds that increase the half-life of acompound in the treated animal. Suitable carriers include, but are notlimited to, polymeric controlled release vehicles, biodegradableimplants, liposomes, bacteria, viruses, oils, esters, and glycols.Preferred controlled release formulations are capable of slowlyreleasing a composition of the present invention into an animal.Suitable controlled release vehicles include, but are not limited to,biocompatible polymers, other polymeric matrices, capsules,microcapsules, microparticles, bolus preparations, osmotic pumps,diffusion devices, liposomes, lipospheres, and transdermal deliverysystems. Other controlled release vehicles of the present inventioninclude liquids that, upon administration to an animal, form a solid ora gel in situ. Preferred controlled release vehicles are biodegradable(i.e., bioerodible).

[0059] In another aspect of the present invention, the present inventionincludes conducting a toxicity test on an animal to determine thetoxicity of a putative regulatory compound. Toxicity tests for putativeregulatory compounds can be performed, for example, on animals after aputative regulatory compound has been determined to have an effect atthe cellular level on signal transduction, such as the regulation ofcellular inflammatory responses. Such toxicity tests are within theskill of the art, and generally involve testing the toxicity of acompound in vivo or in vitro. A suitable method for testing the toxicityof a putative regulatory compound in vivo can involve scientificallycontrolled administration of the putative regulatory compound to anumber of animals and a period of observance in which the effects of thecompound on various aspects of the animal's biological functions (e.g.,occurrence of tissue damage, functioning of organs and death) are noted.Suitable methods for testing the toxicity of a putative regulatorycompound in vitro can involve scientifically controlled administrationof the putative regulatory compound to a cell and subsequent measurementof cell function, cytotoxicity, or cell death. Cell function can bemeasured by any one of a wide range of assays which will be apparent toone of skill in the art, several of which are herein disclosed (e.g.,tyrosine phosphorylation, calcium mobilization and phosphoinositideassays). Methods to measure cytotoxicity are well known in the art andinclude measurement of the ability to reduce chromogenic substrates suchas the tetrazolium-based MTT or sulphorhodamine blue,ATP-bioluminescence assays and fluorescence assays, for example usingthe Fluorescent Green Protein, among many other readily available assays(see, for example, Bellamy, Drugs 44 (5):690-708, 1992, which isincorporated herein by reference in its entirety). Methods to measurecell death include, for example, Coomassie blue staining, acridineorange staining, terminal deoxynucelotidyl transferase (TDT) assays formeasuring DNA fragmentation, neutral red exclusion, and measuringchanges in forward light scattering in a flow cytometer.

[0060] Another aspect of the present invention includes a method toregulate a cellular function selected from the group consisting ofimmunoglobulin heavy chain class switching, cytokine production orinflammatory cell activation, comprising regulating the activity of aprotein including CD40, JNK and/or p38. Regulation of activity of suchprotein can be achieved by sequestering JNK and/or p38 protein in aninactive complex, regulating the ligand binding activity of CD40,regulating the phosphorylation of JNK and/or p38 protein, regulating theinteraction between JNK and JNKK, regulating the ability of JNK toactivate c-Jun, ATF-2, and Ets-1 and other Ets family members,regulating the interaction between p38 and MEK, regulating the abilityof p38 to activate ATF-2, and Ets-1 and other Ets family members,regulating the expression of endogenous and/or heterologous nucleic acidmolecules encoding a CD40, JNK and/or p38 protein, and combinationsthereof.

[0061] Suitable compounds for sequestering a JNK protein in an inactivecomplex, include compounds that mimic the site at which JNK proteininteracts with JNKK, referred to herein as an activation site JNKmimetope. Suitable compounds for sequestering a p38 protein in aninactive complex, include compounds that mimic the site at which p38protein interacts with MEK, referred to herein as an activation site p38mimetope.

[0062] Suitable compounds for regulating the Interaction between JNK andc-Jun, ATF-2, or Ets-1 or other Ets family members comprise JNK targetsite mimetopes. Suitable compounds for regulating the interactionbetween p38 and ATF-2, or Ets-1 or other Ets family members comprise p38target site mimetopes.

[0063] Suitable compounds for regulating the ligand binding activity ofCD40 include CD40 antagonists of extracellular ligands to CD40.

[0064] Other suitable regulatory compounds of the present inventioninclude pseudosubstrates for a regulatory kinase domains of JNK or p38,a JNK kinase domain mimetope, a p38 kinase domain mimetope and a mutatedCD40, JNK or p38 protein. Pseudosubstrates of a JNK kinase domaininclude small organic molecules, peptides or polypeptides that arephosphorylated by a JNK kinase domain in a similar manner as a JNKsubstrate including c-Jun, ATF-2, or Ets-1 or other Ets family members.Similarly, pseudosubstrates of a p38 kinase domain include small organicmolecules, peptides or polypeptides that are phosphorylated by a p38kinase domain in a similar manner as a p38 substrate including ATF-2, orEts-1 or other Ets family members.

[0065] Suitable methods for regulating the expression of endogenousand/or heterologous nucleic acid molecules encoding CD40, JNK and/or p38protein include methods known to those in the art. For example,oligonucleotides for use in, for example, antisense-, triplexformation-, ribozyme- and/or RNA drug-based technologies can be used toreduce expression of endogenous nucleic acid molecules encoding CD40,JNK and/or p38 protein. The present invention, therefore, includes sucholigonucleotides and methods to interfere with the production of CD40,JNK and/or p38 protein by use of one or more of such technologies.Appropriate expression vectors can be developed by those skilled in theart based upon the cell-type being transformed.

[0066] In accordance with the present invention, a “mimetope” refers toany compound that is able to mimic the ability of a regulatory reagentof the present invention. A mimetope can be a peptide that has beenmodified to decrease its susceptibility to degradation but that stillretains regulatory activity. Other examples of mimetopes include, butare not limited to, protein-based compounds, carbohydrate-basedcompounds, lipid-based compounds, nucleic acid-based compounds, naturalorganic compounds, synthetically derived organic compounds,anti-idiotypic antibodies and/or catalytic antibodies, or fragmentsthereof having desired regulatory activity. A mimetope can be obtainedby, for example, screening libraries of natural and synthetic compoundsfor compounds capable of altering the activity of CD40 or JNK, asdisclosed herein. A mimetope can also be obtained by, for example,rational drug design. In a rational drug design procedure, thethree-dimensional structure of a compound of the present invention canbe analyzed by, for example, nuclear magnetic resonance (NMR) or x-raycrystallography. The three-dimensional structure can then be used topredict structures of potential mimetopes by, for example, computermodelling. The predicted mimetope structures can then be produced by,for example, chemical synthesis, recombinant DNA technology, or byisolating a mimetope from a natural source (e.g., plants, animals,bacteria and fungi).

[0067] Another aspect of the present invention comprises administeringto an animal, a therapeutic composition capable of regulating abiological function including immunoglobulin heavy chain classswitching, cytokine production or inflammatory cell activation. Atherapeutic composition of the present invention is particularly usefulfor preventing or treating diseases involving undesired immunoglobulinand/or cytokine production, or inflammatory cell activation. Inparticular, a therapeutic composition is useful for preventing ortreating diseases involving an allergic or autoimmune response.Preferably, a therapeutic composition of the present invention is usedto prevent or treat a disease, including, but not limited to, allergichypersensitivity, asthma, rheumatoid arthritis, systemic lupuserythematosus (SLE), allergic rhinitis, atopic dermatitis and acutebronchopulmonary aspergillosis. A therapeutic composition is preferablyadministered to a cell having CD40 and more preferably to cellsincluding, but not limited to, Pre-B cells, B lymphocytes, cancer cells,fibroblasts, Langerhans cells, epithelial cells monocytes and dendriticcells.

[0068] A variety of therapeutic compositions can be used to perform theregulation method of the present invention. Such therapeuticcompositions include those compounds described in detail herein, inparticular, compounds identified using a method of the presentinvention. A therapeutic composition of the present invention can beformulated in an excipient that the animal to be treated can tolerate.Examples of such excipients include those described in detail above. Inorder to regulate heavy chain class switching in a cell, a therapeuticcomposition of the present invention can be administered in vivo (i.e.,in an animal) or ex vivo (i.e., outside of an animal, such as in tissueculture), in an effective manner such that the composition is capable ofregulating heavy chain class switching.

[0069] An effective administration protocol (i.e., administering atherapeutic composition in an effective manner) comprises suitable doseparameters and modes of administration that result in prevention ortreatment of a disease. Effective dose parameters and modes ofadministration can be determined using methods standard in the art for aparticular disease. Such methods include, for example, determination ofsurvival rates, side effects (i.e., toxicity) and progression orregression of disease. For example, the effectiveness of dose parametersand modes of administration of a therapeutic composition of the presentinvention can be determined by assessing response rates. Such responserates refer to the percentage of treated patients in a population ofpatients that respond with either partial or complete remission.

[0070] In accordance with the present invention, a suitable single dosesize is a dose that is capable of preventing or treating an animal witha disease when administered one or more times over a suitable timeperiod. Doses can vary depending upon the disease being treated. Forexample, in the treatment of hypersensitivity, a suitable single dosecan be dependent upon the nature of the immunogen causing thehypersensitivity.

[0071] It will be obvious to one of skill in the art that the number ofdoses administered to an animal is dependent upon the extent of thedisease and the response of an individual patient to the treatment. Forexample, in the case of allergic responses, the immunogenicity of acompound may require more doses than a less immunogenic compound. Thus,it is within the scope of the present invention that a suitable numberof doses, as well as the time periods between administration, includesany number required to cause treat a disease.

[0072] Therapeutic compositions can be administered directly to a cellin vivo or ex vivo or systemically. Preferred methods of systemicadministration, include intravenous injection, aerosol, oral andpercutaneous (topical) delivery. Intravenous injections can be performedusing methods standard in the art. Aerosol delivery can also beperformed using methods standard in the art (see, for example, Striblinget al., Proc. Natl. Acad. Sci. USA 189:11277-11281, 1992, which isincorporated herein by reference in its entirety). Oral delivery can beperformed by complexing a therapeutic composition of the presentinvention to a carrier capable of withstanding degradation by digestiveenzymes in the gut of an animal. Examples of such carriers, includeplastic capsules or tablets, such as those known in the art. Topicaldelivery can be performed by mixing a therapeutic composition of thepresent invention with a lipophilic reagent (e.g., DMSO) that is capableof passing into the skin.

[0073] The following examples are provided for the purposes ofillustration and are not intended to limit the scope of the presentinvention.

EXAMPLES Example 1

[0074] This example describes the activation of ERK by ligation ofsurface IgM but not CD40.

[0075] The human Burkitt's lymphoma cell line Ramos was obtained fromthe American Type Culture Collection (Rockville, Md.), and cells weremaintained in RPMI-1640 supplemented with 50 units/mlpenicillin-streptomycin, 2 mM glutamine, and 10% FCS. Exponentiallygrowing cells were used in all experiments.

[0076] A. Immunoblot Assays Using Ramos Cells

[0077] About 1×10⁶ Ramos cells were separately treated with 10 μg/mlanti-IgM F(ab′)2 goat anti-human IgM antibody (obtained from Zymed, SanFrancisco, Calif.) or 5 μg/ml anti-CD40 antibody (G28-5; obtained fromDr. E. A. Clark, Washington University, Seattle, Wash.) for 0, 1, 5, 15,30 or 60 min. Ramos cells were also treated with phorbol 12-myristate13-acetate (PMA; obtained from Sigma, St Louis, Mo.). Immunoblotanalysis was performed on each of the samples using monoclonal anti-ERK2antibody in the following method. The treated cells were lysed in 100 μlof lysis buffer (25 mM Tris-HCl, pH 7.6, 50 mM NaCl, 0.5% Nadeoxycholate, 2% NP-40, 0.2% SDS, 1 mM PMSF, 50 μg/ml aprotinin, 50 μMleupeptin). Lysates were centrifuged for 10 min at 14,000 rpm in anEppendorf microcentrifuge, 90 μl of supernatants were mixed with 30 μlof 4×Laemmli sample buffer. Samples were boiled for 5 min. Twenty μl ofprepared samples were electrophoresed through a 12% SDS-PAGE gel, andproteins were transferred to nitrocellulose membranes. Membranes wereincubated in blocking buffer (25 mM Tris-HCl, pH 8.0, 125 mM NaCl, 0.1%Tween 20, 2% BSA, 0.1% NaN₃) at 4° C. overnight, then monoclonal mouseanti-ERK2 antibody (1:2000; obtained from Upstate BiotechnologyIncorporated, Lake Placid, N.Y.) was added to the blocking buffer, andblots were incubated for an additional 2 hours at room temperature. Theblots were washed three times in TBST (25 mM Tris-HCl, pH 8.0, 125 mMNaCl, 0.025% Tween 20) and incubated with AP-conjugated goat anti-mouseIg (1:10000 in TBST; obtained from Promega, Madison, Wis.) for 1 hour atroom temperature. The blots were washed three times in TBST anddeveloped with the colorogenic substrates BCIP and NBT (Promegaprotoblot AP system).

[0078] The results from the immunoblot indicated that the untreatedsamples contained a single band (42 kD) reactive with anti-ERK2 antibody(lane 0′). In samples treated with PMA (100 ng/ml) or anti-IgM for 20min, a second band with immunoreactivity to anti-ERK2 antibody appeared(lane PMA and anti-IgM lane 1′-60′). This lower mobility form representsthe activated form due to phosphorylation. The samples treated withanti-CD40 demonstrated only a single band throughout the time course,thus indicating no lower mobility shift in p42^(erk2). These dataindicate that anti-IgM antibody activates p42^(erk2) but anti-CD40antibody fails to activate p42^(erk2).

[0079] B. Immunoblot Assays Using Tonsillar Cells

[0080] The procedure of step A was repeated except using lysates fromfreshly isolated tonsillar B cells (prepared from tonsils as describedin Takase et al., J. Cell Physiol. 162:246-255, 1995). Followingtreatment with 5 μg/ml anti-CD40 for the indicated times also showed noshift in ERK2 mobility.

[0081] B. ERK Kinase Assay

[0082] Kinase activity was evaluated using EGFRF₆₆₂₋₆₈₁ peptide as asubstrate as described previously (Franklin et al., J. Immunol.153:4890-4898, 1994). Following stimulation, 10⁶ cells were lysed in 75μl of lysis buffer (70 mM β-glycerophosphate, pH 7.2, 100 mM Na₃VO₄, 2mM MgCl₂, 1 mM EGTA, 0.5% Triton X-100, 5 μg/ml leupeptin, 2 μg/mlaprotinin and 1 mM DTT) and placed on ice for 15 min. Cell lysates werecentrifuged at 14,000 rpm for 10 min and 20 μl of supernatant wereremoved and mixed with 20 μl of 2×kinase buffer (50 mMβ-glycerophosphate, pH 7.2, 100 mM Na₃VO₄, 20 mM MgCl₂, 50 mg/ml IP-20,1 mM EGTA, 400 μM EGFR₆₆₂₋₆₈₁ peptide, 200 μM ATP and 0.225mCi/ml[γ-³²P] ATP [ICN Biochemicals, Costa Mesa, Calif.]). After 15 minat 30° C., 10 μl of 250 mM EDTA was added, and 45 μl of the reactionmixture was spotted onto P-81 phosphocellulose paper (Whatman, Clifton,N.J.). The papers were washed four times (5 min each) in 400 ml of 75 mMphosphoric acid and then radioactivity bound to the filter paper wasdetermined by liquid scintillation counting. The assay system containedboth EGTA (1 mM) and IP-20 (25 mg/ml), reagents that should effectivelyinhibit PKC, calcium/calmodulin, and cAMP-dependent kinases.

[0083] The results (shown in FIG. 1) indicate the kinase activity ofsamples treated with 100 ng/ml PMA for 20 min and 10 μg/ml anti-IgM for5 min were about 30,000 counts per minute compared with samples fromunstimulated (control) cells (about 10,000 cpm) or cells treated with 1min (about 15,000 cpm), 5 min (about 17,000 cpm), or 10 μg/ml anti-CD40for 20 min (about 15,000 cpm). The data represent incorporation of ³²p(±SD) from separately prepared duplicate samples from two independentexperiments. Statistically significant differences from untreated (0′)samples are represented by an asterisk (*) (p<0.05).

[0084] The results confirm the results obtained in the immunoblotexperiments. Activation of p42^(erk2) by anti-IgM was confirmed byincreases in EGFR₆₆₂₋₆₈₁ peptide phosphorylation. In contrast, theaddition of anti-CD40 antibody at concentrations up to 10 μg/ml, failedto activate p42^(erk2) in Ramos cells. Furthermore, we confirmed thatanti-CD40 failed to activate p42^(erk2) in freshly isolated tonsillar Bcells.

Example 2

[0085] This example demonstrates that c-Jun kinase is activated byanti-CD40 antibody and soluble gp39 but not by anti-IgM antibody.

[0086] JNK activity was measured by solid-phase kinase assay usingGST-c-Jun (1-79) as a substrate following treatment with anti-IgMantibody or anti-CD40 antibody (G28-5) in Ramos cells (three independentexperiments) and tonsillar B cells (two independent experiments).GST-c-Jun (1-79) fusion protein was purified from bacterial lysatesusing GSH-Sepharose beads (Pharmacia Biotech, Uppsala, Sweden) at roomtemperature with gentle rocking using the method described inGalcheva-Gargova et al. (Science 265:806-808, 1994). Followingstimulation, 3×10⁶ cells were lysed in lysis buffer (20 mM Tris-HCl, pH7.6, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 0.5% NP-40, 2 mM Na₃VO₄, 1 mMDTT, 1 mM PMSF, 20 μg/ml aprotinin, 5 μg/ml leupeptin). The lysates weremixed with 10 μl of GST-c-Jun (1-79) coupled to GSH-Sepharose beads. Themixture was rotated at 4° C. for 3 hr in a microcentrifuge tube andpelleted by centrifugation at 14,000 rpm for 5 min. The pelleted beadswere washed 2 times in lysis buffer and once in kinase buffer (20 mMHepes, pH 7.5, 20 mM β-glycerophosphate, 10 mM MgCl₂, 1 mM DTT, 50 mMNa₃VO₄, 10 mM p-nitrophenyl phosphate), and then resuspended in 40 μl ofkinase buffer containing 10 μCi of [γ-³²p]ATP. After 20 min at 30° C.,the reaction was terminated by adding 4×Laemmli sample buffer andboiling for 3 min. Samples were resolved by 12% SDS-PAGE and subjectedto autoradiography. Phosphate incorporation was determined byPhosphorImager (Molecular Dynamics, Sunnyvale, Calif.). The level of ³²Pincorporation (±SD) into the substrate is illustrated as the ratio ofJNK activity to that of untreated samples.

[0087] Results from a time course of JNK activation after treatment ofRamos cell with 1 μg/ml anti-CD40 antibody indicated a rapid and markedincrease in JNK activation within 1 min of treatment with 1 μg/mlanti-CD40 antibody (FIG. 2), reached peak levels within 15 min and thenbegan to decline by 30-60 min. Statistically significant differencesfrom untreated (040 ) samples are represented by an asterisk (*)(p<0.05).

[0088] Results from a dose response study of anti-CD40antibody-activated JNK in Ramos cells treated with variousconcentrations of anti-CD40 antibody for 15 min. indicated that, in thepresence of 0.5 μg/ml anti-CD40 antibody, levels of ³²P incorporationwere five-fold higher than control samples (FIG. 3). JNK activityincreased in a dose dependent fashion with peak levels (aboutseven-fold) observed at a concentration of 2-5 μg/ml antibody.Statistically significant differences from untreated (0′) samples arerepresented by an asterisk (*) (p<0.05).

[0089] A dose-dependent response to anti-CD40 antibody was also detectedin tonsillar B cells (FIG. 4). Throughout the dose-response curve lowerlevels of activation were observed in tonsillar B cells relative toRamos cells, but both cell types clearly respond to CD40 ligation with asignificant JNK activation.

[0090] Results from the treatment of Ramos cells for 15 min withdilutions of culture supernatants containing soluble gp39 (prepared asdescribed in Hollenbaugh et al., EMBO J. 11:4313-4321, 1992) indicatedthat recombinant soluble gp39 also activated JNK in a dose-dependentfashion in Ramos cells (FIG. 5) and tonsillar B cells.

[0091] Results from blocking studies using dilutions of culturesupernatants containing soluble gp39 that were pre-incubated withanti-gp39 antibody (2 μg/ml; mAb39-1.106; described in Bajorath et al.,Biochemistry 34:1833-1844, 1995) for 5 min prior to addition to Ramoscells indicated that anti-gp39 antibody prevented activation of JNK bysoluble gp39. JNK activation by UV irradiation was unaffected by thepresence of the antibody. The anti-gp39 antibody failed to blockUV-irradiation-induced activation of JNK.

[0092] Results from a time course cross-linking study using anti-IgMantibody indicated that JNK activity was not increased following surfaceIgM crosslinking even in the presence of 10 μg/ml anti-IgM antibody inRamos cells (FIG. 6) and tonsillar B cells (FIG. 7).

[0093] Taken together, the results demonstrate that JNK is activated byanti-CD40 antibody but not anti-IgM antibody, indicating that anti-CD40antibody activates JNK through a different signaling pathway than thatwhich mediates ERK activation by anti-IgM antibody.

Example 3

[0094] This example demonstrates that anti-CD40 antibody activates JNKthrough a Ras-independent pathway.

[0095] Metabolically labeled (³²P)Ramos cells were untreated (control)or treated with 10 μg/ml anti-IgM or 5 μg/ml anti-CD40 for 1, 5 and 10min, respectively. Ras was immunoprecipitated using the Y13-259 anti-Rasantibody, and radioactive GTP and GDP bound to Ras was measured asfollows. Cells (10⁷ cells) were labeled with ³²P-orthophosphate for 16hr, and then stimulated. Ras was immunoprecipitated using the Y13-259anti-Ras antibody, and GTP was separated from GDP by thin layerchromatography as described (Downward et al., Nature 346:719-723, 1990).The radiolabeled nucleotides were visualized by autoradiography.Radioactivity was quantitated with a PhosphorImager and theGTP/GTP+(1.5) GDP ratios were calculated. The data were quantitated byPhosphorImager, and shown are the GTP/GDP+(1.5) GDP ratios (in percent)for each condition. The results represent three separate experiments.Statistically significant differences from untreated (0′) samples arerepresented by an asterisk (*) (p<0.05).

[0096] The results, shown in FIG. 8, indicate that treatment withanti-IgM antibody activated Ras. Anti-CD40 antibody treatment, however,failed to activate Ras at concentrations that were effective in JNKactivation (described in Example 2). The results demonstrate that thesignals transduced following CD40 engagement lead to JNK activationthrough a pathway that does not involve Ras activation.

Example 4

[0097] This example demonstrates that Raf-1 does not participate in theCD40-activated JNK pathway.

[0098] Ramos cells were untreated or treated with 100 ng/ml PMA, 10μg/ml anti-IgM or 5 μg/ml anti-CD40 for 1, 2.5, 5, 10 or 20 min. Raf-1was immunoprecipitated and a kinase assay was performed using thefollowing method. Cells (10⁷) were stimulated in RPMI 1640 medium, andthen lysed in RIPA (10 mM sodium phosphate, pH 7.0, 150 mM NaCl, 2 mMEDTA, 1% sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS, 1% aprotinin,50 mM NaF, 200 mM Na₃VO₄, 0.1% 2-mercaptoethanol, 1 mM PMSF). Thelysates were precleared by protein A-Sepharose beads for 30 min at 4° C.A purified polyclonal anti-Raf-1 antibody was added to the lysates(1:100; obtained from Santa Cruz Biotechnology, Santa Cruz, Calif.) andincubated for 90 min at 4° C. The immunocomplexes were collected byprotein A-Sepharose beads. The beads were then washed 3 times in RIPAand 3 times in a buffer containing 10 mM PIPES, pH 7.0, 100 mM NaCl, 2μg/ml aprotinin. A kinase mixture (40 μl) containing 10 mM PIPES, pH7.0, 100 mM NaCl, 5 mM MnCl₂, 2 μg/ml aprotinin, 30 μCi of [γ-³²P]ATPand 100-200 ng of catalytically inactive MEK (KMMEK) was added to thebeads. KMMEK was expressed and purified as described (Gardner et al.,Methods Enzymol. 238:258-270, 1994). The samples were incubated for 30min at 30° C. The kinase reaction was stopped by addition of 4×Laemmlisample buffer and boiling for 3 min. The proteins were resolved on 10%SDS-PAGE and transferred to a nitrocellulose membrane. The membrane wasprobed using the same anti-Raf-1 antibody and visualized as describedabove and subjected to autoradiography.

[0099] The results indicate that the levels of KMMEK phosphorylationfollowing treatment with 5 μg/ml anti-CD40 antibody were not differentthan control samples, whereas cells treated with anti-IgM antibodyresulted in increased KMMEK phosphorylation. To verify similar loadingof immunoprecipitated Raf-1, an immunoblot was concomitantly performedusing the same antibody as was used for the immunoprecipitates. TheRaf-1 mobility shifts were consistent with the increased levels ofkinase activity measured using KMMEK. The magnitude of anti-IgM antibodyactivation of Raf-1 was sufficient to activate ERK2 similarly as PMA.Raf-1, which is an efficient activator of the ERK pathway, is notmeasurably activated during JNK activation in response to CD40 ligation.

Example 5

[0100] This example demonstrates that anti-CD40 antibody activates MEKKprotein.

[0101] Ramos cells were treated with 2 μg/ml anti-CD40 antibody for 0,0.5, 1, 2.5, 5 or 10 min. MEKK was immunoprecipitated and a kinase assaywas performed using the following methods. Following stimulation, 5×10⁶cells were lysed in 400 μl of extraction buffer (1% Triton X-100, 10 mMTris-HCl [pH 7.4], 5 mM EDTA, 50 mM NaCl, 50 mM NaF, 0.1% bovine serumalbumin, aprotinin [20 μg/ml], 1 mM PMSF, and 2 mM Na₃VO₄). The lysateswere centrifuged for 10 min at 14,000 rpm and pellets were discarded.The supernatants were incubated with the rabbit MEK kinase (MEKK)antisera (1:100 dilution) raised against the MEKK NH2-terminal fusionprotein (described in Lange-Carter et al., Methods Enzymol. 255:290-301,1995) for 2 hr at 4° C. The immune complexes were collected by proteinA-Sepharose beads. The beads were then washed twice in RIPA buffer andthree times in a buffer containing 10 mM PIPES, pH 7.0, 100 mM NaCl, 2μg/ml aprotinin. A kinase mixture (40 μl) containing 10 mM PIPES, pH7.0, 100 mM NaCl, 5 mM MnCl₂, 2 μg/ml aprotinin, 30 μCi [γ-³²P]ATP and0.5 μl of recombinant JNK activating protein kinase (JNKK; described inLin et al., Science 268:286-290, 1995) as a substrate was added to thebeads. The samples were incubated for 30 min at 30° C. The kinasereaction was stopped by addition of4×Laemmli sample buffer and boilingfor 3 min. The proteins were resolved on 10% SDS-PAGE, transferred to anitrocellulose membrane and subjected to autoradiography. Phosphateincorporation was quantitated by PhosphorImager.

[0102] The results of ³²P incorporation into JNKK are illustrated inFIG. 9 as the ratio of MEKK activity of treated to that of untreatedsamples. Statistically significant differences from untreated (0′)samples are represented by an asterisk (*) (p<0.05). MEKK was activatedrapidly, reaching maximal stimulation by 30 sec after anti-CD40 antibodytreatment, and then decreased gradually with time. Immunoblots of theimmunoprecipitated MEKK with the anti-MEKK polyclonal antibody that wereused for immunoprecipitation revealed similar amounts of a 98 kD MEKKprotein for each time point. These data indicate that an MEKK is presentin B-lymphoblastoid cells which regulates the JNK pathway and isactivated in response to CD40 ligation.

Example 6

[0103] This example demonstrates that anti-CD40 antibody rescuesapoptosis induced by anti-IgM antibody.

[0104] Ramos cells were untreated (control) or treated with 10 μg/mlanti-IgM antibody or 2 μg/ml anti-CD40 antibody; co-stimulation of cellsconsisted of a 30 min preincubation with anti-CD40 antibody followed byan incubation with anti-IgM antibody. After 18 hr culture, DNA breaksderived from anti-IgM induced apoptosis were evaluated using an in situTdT assay using the method as follows. For detection of DNA strandbreaks in individual cells, an in situ terminal deoxynucleotidyltransferase (TdT) assay was employed based on the method of Gorczyca etal. (Cancer Res. 53:3186-3192, 1993) with minor modifications. Cellswere treated for 18 hr as indicated. About 10⁶ cultured cells werewashed in PBS and suspended in 500 μl of PBS. Paraform (4%, 170 μl) wasadded and the mixture was stored on ice for 15 min. Cells were thenwashed in cold PBS and fixed with 70% ethanol at −20° C. for an hour.Following washing in cold PBS, the cells were resuspended in TdTreaction buffer (0.1 M potassium cacodylate, pH 7.2, 2 mM CoCl₂, 0.2 mMDTT, 20 U TdT, 2 nmol fluorescein-dUTP, 10 mg/ml BSA). After 30 min at37° C., cells were washed once in 0.2% BSA/PBS and fluoresce stainingwas evaluated on an EPICS Profile (Coulter, Hialeah, Fla.).

[0105] p42^(erk2) (5 min following treatment) and JNK activation (15 minfollowing treatment) were evaluated under identical conditions asdescribed in Example 1. The level of ³²P incorporation (±SD) from twoindependent experiments was evaluated by PhosphorImager, and thenplotted as the ratio of JNK activation to that of untreated samples.Statistically significant differences from untreated samples arerepresented by an asterisk (*) (p<0.05).

[0106] The results indicate that DNA breaks were detected in 64.2% ofRamos cells, 18 hr following treatment with 10 μg/ml anti-IgM antibody,whereas there was no shift in fluorescence intensity in control(untreated) cells or in cells treated with 2 μg/ml anti-CD40 antibody.In the presence of 2 μg/ml anti-CD40 antibody preincubated for 30 minprior to the addition of anti-IgM antibody, however, DNA breaks inducedby anti-IgM antibody were reduced to 3.5% of the cells. Under identicalconditions, an immunoblot using anti-ERK2 monoclonal antibody indicatedthe mobility shift in p42^(erk2) protein, 5 min following treatment with10 μg/ml anti-IgM antibody in the presence of 2 μg/ml anti-CD40 antibodypreincubated for 30 min. In addition, JNK activity, measured by solidphase kinase assay using GST-c-Jun fusion protein, was increased 15 minfollowing treatment with anti-CD40 antibody in the presence of anti-IgMantibody. Thus, CD40 ligation does not effect p42^(erk2) activation bysIgM and sIgM ligation does not effect CD40 activation of JNK.

Example 7

[0107] This example describes the activation of p38 by CD40 ligation.

[0108] Neutrophils isolated by the plasma percoll method as previouslydescribed (Haslett, C., Am. J. Pathol. 119, 101-110; 1985) wereresuspended at about 25×10⁶ cells/ml in KRPD containing 0.1% Human HeatInactivated Platelet-Poor Plasma, 1 mM PMSF, 10, μg/ml leupeptin and 10μg/ml aprotinin. About 25×10⁶ PMN were preincubated for 30 minutes at37° C., then stimulated with 100 ng/ml LPS for varying time intervalsand reactions terminated by a 20 second centrifugation at 15,000 rpm.Cell pellets were lysed with 500 μl cold RIPA (50 mM Tris pH=7.2, 150 mMNaCl, 1.1% SDS, 0.1% sodium deoxycholate, 1% Triton-X-100, 10 mM sodiumpyrophosphate, 25 mM M glycerophosphate, 1 mM sodium orthovanadate and2.1 μg/ml aprotinin), and centrifuged at 15,000 for 10 minutes at 4° C.Triton soluble lysates were initial precleared with Protein A Sepharosefor 30 minutes at 4° C., followed by Protein A Sepharoseimmunoprecipitation with 5 μl/sample polyclonal antibodies specific forp38 and 15 μl of bead suspension, for 120 minutes at 4° C. Beads werewashed once in RIPA and twice in PAN (10 mM Pipes, 100 mM NaCl, pH=7.0,and 21 μg/ml aprotinin). Beads were resuspended 25 μl kinase mixcontaining 20 mM Hepes, pH=7.6, 200 mM MgCl₂, 20 μM ATP, 20 μCi[³²P]γ-ATP, 2 mM dTT, 100 μM sodium orthovanadate, 25 mMB-glycerophosphate (pH=7.2) and a peptide comprising amino acids 1-110of ATF-2. The samples were incubated for 15 minutes at 30° C. withfrequent mixing. Reactions were terminated with 2×Laemelli buffer andafter boiling, proteins were separated by SDS-PAGE, with quantificationof activity by autoradiography and phosphorimaging, and qualitativeanalysis of enzyme presence and phosphorylation by Western Blotting.

[0109] The results are shown in FIG. 10 and indicate that p38 MAP kinaseis activated in neutrophils by lipopolysaccharide.

[0110] While various embodiments of the present invention have beendescribed in detail, it is apparent that modifications and adaptationsof those embodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims:

What is claimed is:
 1. A method to identify a compound that controlsCD40 regulation of JNK activity in a cell, comprising: (a) contacting acell with a putative regulatory compound, wherein the cell includes aCD40 protein and a Jun kinase protein; and (b) assessing the ability ofsaid putative regulatory compound to regulate the activity of said Junkinase.
 2. The method of claim 1 , wherein said assessment stepcomprises determining the ability of said JUN kinase protein to regulatethe activity of a protein selected from the group consisting of c-Jun,ATF-2, Ets-1 and other Ets family members activity in said cell.
 3. Themethod of claim 1 , wherein said method further comprises stimulatingsaid cell, prior to said assessing step, with a ligand of CD40.
 4. Themethod of claim 1 , wherein said method further comprises stimulatingsaid cell, prior to said assessment step, with a stimulatory compoundselected from the group consisting of soluble gp39, membrane-bound gp39and an antibody that binds specifically to CD40.
 5. The method of claim1 , wherein said cell is selected from the group consisting of amammalian, an invertebrate, a plant, an insect, a fungal, a yeast and abacterial cell.
 6. The method of claim 1 , wherein said cell is selectedfrom the group consisting of a mammalian, an amphibian and a yeast cell.7. The method of claim 1 , wherein said cell is selected from the groupconsisting of a primate, a mouse and a rat cell.
 8. The method of claim1 , wherein said cell is selected from the group consisting of Pre-Bcells, B lymphocytes, cancer cells, fibroblasts, Langerhans cells,epithelial cells monocytes and dendritic cells.
 9. The method of claim 1, wherein said putative regulatory compound is selected from the groupconsisting of a protein-based compound, a carbohydrate-based compound, alipid-based compound, a nucleic acid-based compound, a natural organiccompound, a synthetically derived organic compound, an anti-idiotypicantibody and/or catalytic antibody, or fragments thereof.
 10. The methodof claim 1 , wherein said putative regulatory compound is selected fromthe group consisting of a small organic molecule, a peptide and apolypeptide.
 11. A method to identify a compound that controls CD40regulation of p38 activity in a cell, comprising: (a) contacting a cellwith a putative regulatory compound, wherein the cell includes CD40protein and p38 protein; and (b) assessing the ability of said putativeregulatory compound to regulate the activity of said p38 protein. 12.The method of claim 11 , wherein said assessment step comprisesdetermining the ability of said p38 protein to regulate the activity ofa protein selected from the group consisting of ATF-2, Ets-1 and otherEts family members activity in said cell.
 13. The method of claim 11 ,wherein said method further comprises stimulating said cell, prior tosaid assessing step, with a ligand of CD40.
 14. The method of claim 11 ,wherein said method further comprises stimulating said cell, prior tosaid assessment step, with a stimulatory compound selected from thegroup consisting of soluble gp39, membrane-bound gp39 and an antibodythat binds specifically to CD40.
 15. The method of claim 11 , whereinsaid cell is selected from the group consisting of Pre-B cells, Blymphocytes, cancer cells, fibroblasts, Langerhans cells, epithelialcells monocytes and dendritic cells.
 16. The method of claim 11 ,wherein said putative regulatory compound is selected from the groupconsisting of a small organic molecule, a peptide and a polypeptide. 17.A regulatory compound identified by said compound's ability to regulatea biological function selected from the group consisting ofimmunoglobulin heavy chain class switching, cytokine production andinflammatory cell activation, said compound being capable of penetratingthe plasma membrane of a cell and of inhibiting the ability of CD40protein to regulate JNK protein activity in said cell.
 18. The compoundof claim 17 , wherein said compound is capable of regulating theactivity of a protein selected from the group consisting of CD40 and Junkinase protein.
 19. The compound of claim 17 , wherein said regulationinvolves a step selected from the group consisting of sequestering JNKprotein in an inactive complex, regulating the ligand binding activityof CD40, regulating MEKK activity, regulating the phosphorylation of JNKprotein, regulating the interaction between JNK and JNKK, regulating theability of JNK to activate c-Jun, ATF-2, and Ets-1 and other Ets familymembers, regulating the, expression of endogenous and/or heterologousnucleic acid molecules encoding CD40 or JNK protein, and combinationsthereof.
 20. A regulatory compound capable of penetrating the plasmamembrane of a cell and of inhibiting the ability of CD40 protein toregulate p38 protein activity in said cell, said compound able toregulate a biological function selected from the group consisting ofimmunoglobulin heavy chain class switching, cytokine production andinflammatory cell activation.
 21. The compound of claim 20 , whereinsaid compound is capable of regulating the activity of a proteinselected from the group consisting of CD40 and p38 protein.
 22. Thecompound of claim 20 , wherein said regulation involves a step selectedfrom the group consisting of sequestering p38 protein in an inactivecomplex, regulating the ligand binding activity of CD40, regulating MEKKactivity, regulating the phosphorylation of p38 protein, regulating theinteraction between p38 and MEK, regulating the ability of p38 toactivate ATF-2, and Ets-1 and other Ets family members, regulating theexpression of endogenous and/or heterologous nucleic acid moleculesencoding CD40 or p38 protein, and combinations thereof.
 23. A method toinhibit immunoglobulin heavy chain class switching, comprisinginhibiting the activity of a protein selected from the group consistingof Jun kinase protein and p38 protein.
 24. The method of claim 23 ,wherein said activity being inhibited is selected from the groupconsisting of sequestering JNK or p38 protein in an inactive complex,regulating the ligand binding activity of CD40, regulating MEKKactivity, regulating the phosphorylation of JNK or p38 protein,regulating the interaction between JNK and JNKK, regulating the abilityof JNK to activate c-Jun, ATF-2, and Ets-1 and other Ets family members,regulating the interaction between p38 and MEK, regulating the abilityof p38 to activate ATF-2, and Ets-1 and other Ets family members,regulating the expression of endogenous or heterologous nucleic acidmolecules encoding a CD40, JNK or p38 protein, and combinations thereof.25. The method of claim 24 , wherein said activity is inhibited using acompound selected from the group consisting of a Jun kinase activationsite mimetope, a Jun kinase target site mimetope, a Jun kinase kinasedomain mimetope, a mutated Jun kinase protein, a Jun kinasepseudosubstrate, a p38 activation site mimetope, a p38 target sitemimetope, a p38 kinase domain mimetope, a mutated p38 protein, a p38pseudosubstrate, a mutated CD40 protein, a CD40 antagonist, an antisenseoligonucleotide, a ribozyme and an expression plasmid encoding CD40 orJun kinase.
 26. A method to inhibit cytokine production by a cell havingCD40, comprising inhibiting the activity of a protein selected from thegroup consisting of Jun kinase protein and p38 protein.
 27. The methodof claim 26 , wherein said activity is inhibited using a compoundselected from the group consisting of a Jun kinase activation sitemimetope, a Jun kinase target site mimetope, a Jun kinase kinase domainmimetope, a mutated Jun kinase protein, a Jun kinase pseudosubstrate, ap38 activation site mimetope, a p38 target site mimetope, a p38 kinasedomain mimetope, a mutated p38 protein, a p38 pseudosubstrate, a mutatedCD40 protein, a CD40 antagonist, an antisense oligonucleotide, aribozyme and an expression plasmid encoding CD40 or Jun kinase.
 28. Amethod to treat an animal with a disease selected from the groupconsisting of a disease involving an allergic response and a diseaseinvolving an autoimmune disease, said method comprising administering toan animal an effective amount of a therapeutic composition comprising acompound that controls CD40 regulation of the activity of a proteinselected from the group consisting of Jun kinase and p38 protein. 29.The method of claim 28 , wherein said disease involving an allergicresponse is selected from the group consisting of asthma, allergicrhinitis, allergic hypersensitivity, atopic dermatitis and acutebronchopulmonary aspergillosis.
 30. The method of claim 28 , whereinsaid disease involving an autoimmune disease is selected from the groupconsisting of rheumatoid arthritis and systemic lupus erythematosus. 31.A kit to identify compounds that controls CD40 regulation of JNKactivity in a cell, said kit comprising: (a) a cell comprising a CD40protein and a Jun kinase protein; and (b) a means for detectingregulation of said Jun kinase protein.
 32. The kit of claim 32 , whereinsaid cell is selected from the group consisting of Pre-B cells, Blymphocytes, cancer cells, fibroblasts, Langerhans cells, epithelialcells monocytes and dendritic cells.
 33. A kit to identify compoundsthat controls CD40 regulation of p38 activity in a cell, said kitcomprising: (a) a cell comprising CD40 protein and p38 protein; and (b)a means for detecting regulation of said p38 protein.
 34. The kit ofclaim 33 , wherein said cell is selected from the group consisting ofPre-B cells, B lymphocytes, cancer cells, fibroblasts, Langerhans cells,epithelial cells monocytes and dendritic cells.